The cellular mechanisms involved in adrenocorticotropin (ACTH) release were studied in a tumor cell line of the mouse anterior pituitary (AtT-20/D16-16). Forskolin, and effective activator of adenylate cyclase increases cAMP accumulation, cAMP-dependent protein kinase activity and ACTH release with equal potency. The phosphorylation of several endogenous proteins is altered by forskolin acting through cAMP-dependent protein kinase. These data support a role for cAMP as a second messenger in the receptor-mediated release of ACTH. Somatostatin (SRIF) is a potent inhibitor of ACTH release from AtT-20 cells. This peptide blocks hormone and forskolin-stimulated cyclic AMP formation and ACTH release by activating a guanine nucleotide inhibitory protein (Ni). This proposal is supported by experiments employing pertussis toxin. This agent catalyzes the ADP-ribosylation of Niin AtT-20 cell membranes. Pertussis toxin blocks the ability of SRIF to inhibit corticotropin releasing factor (CRF) and forskolin-stimulated cAMP accumulation and ACTH release by inactivating Ni. SRIF receptors can become desensitized on AtT-20 cells. Pretreatment with SRIF reduces the subsequent ability of SRIF to inhibit CRF and forskolin-stimulated cAMP accumulation and ACTH. This desensitization may involve an uncoupling of Ni and SRIF receptors.